Identification and regulation of the juvenile hormone esterase gene in the Colorado potato beetle

A number of important physiological processes in insects is controlled by the titer of juvenile hormone (JH). The juvenile (larval) stage is maintained at a high JH titer, whereas the onset of metamorphosis is induced by a low JH titer. Reproduction by adults requires often a high JH titer. Through synchronization of the reproductive phase with favorable environmental conditions, insects are able to adapt to adverse conditions and to enhance the chance for survival. The Colorado potato beetle uses daylength (photoperiod) as key stimulus for seasonal adaptation.In temperate areas the beetle hibernates in the soil and development is arrested. If the adult beetle is exposed to short-day conditions it digs into the soil after 11-12 days, which marks the onset of diapause. This photoperiodic effect is mediated by JH. Long-day conditions result in a high JH titer leading to reproduction. Short-day conditions, however, result in a low JH titer leading to diapause. The JH titer in the hemolymph is the result on the one hand by JH synthesis in the corpora allata and on the other hand by JH degradation in the hemolymph. Hydrolysis of the JH ester by JH esterase (JHE) is the main JH degradation pathway in the beetle. The highest JHE activity occurs in the hemolymph (Chapter 1).Surprisingly, the JHE from the Colorado potato beetle (Coleoptera) was found to be a dimer, consisting of two subunits of 57 kDa, whereas lepidopteran JHEs were characterized as monomers. The fact that the beetle JHE occurs as a dimer, is also the reason why the enzyme could be separated from monomeric general carboxyl esterases by nondenaturing polyacrylamide gel electrophoresis (PAGE). After PAGE purification, narrow-range isoelectric focusing and SDS-PAGE were employed to purify JHE from hemolymph of the last larval instar of the Colorado potato beetle. Two forms were found with isoelectric points of 5.5 and 5.6, respectively (Chapter 2).To clone the encoding cDNA, the purified JHE was subjected to endoproteinase Lys-C digestion. Based on the amino acid sequence of the separated peptides, degenerate primers were designed to perform RT-PCR in order to clone an internal fragment of the encoding cDNA. The 3'-end was found by screening a cDNA library and the 5'-end by using the 5'RACE technique. The size of the transcript was 1.7 kilobase. The deduced amino acid sequence (515 residues) of the putative JHE cDNA showed limited homology to some functional peptide motifs of other insect esterases (Chapter 3).RT-PCR on mRNA and analysis of genomic DNA provided evidence for the existence of two JHE-related genes, JHE.A and JHE.B. Both are intronless in the coding regions. JHE.A with a predicted pI of 5.5 probably codes for hemolymph JHE. The function of JHE.B with a predicted pI of 6.9 is unknown yet, but it may code for cytosolic JHE in the fat body. Both genes lack a conserved motif with a catalytic serine, typical for serine esterases. It was hypothesized that dimerization of the 57 kDa subunits can generate a catalytic site for enzyme activity (Chapter 4).In the beetle, the JHE transcript was mainly synthesized during the feeding stage of the last larval instar as well as in pre-diapausing animals (short-day adults). At these stages the JH titer is low and induces pupation and diapause, respectively. JHE activity in the hemolymph correlated with JHE-mRNA levels in the fat body during the development of the beetle. This observation is a strong indication that the probe used to follow JHE-mRNA levels indeed codes for hemolymph JHE. Also after photoperiodic and JH analog treatment the positive correlation was found between JHE-mRNA levels and JHE activity.JH analog (pyriproxifen) application to early 4th-instar larvae resulted in a negative feedback as JHE synthesis was enhanced to maintain the JH titer low. In contrast, if the JH analog was applied to early short-day adult beetles, JHE gene expression was suppressed to reprogram the beetles. Instead of preparation for diapause with a low JH titer and a high JHE level, the treated beetles showed reproductive development with a low JHE level to maintain a high JH titer (Chapter 5).The putative JHE cDNAs, JHE.A and JHE.B, were expressed in a baculovirus-insect cell system. The JHE-specific transcripts were clearly present in the Spodoptera frugiperda cells, as detected by Northern hybridization. Also a 57 kDa protein was synthesized, as visualized by Coomassie Brilliant Blue staining, although it was at a low level. However, JHE enzyme activity could not be detected. During translation or post-translational modification, other unknown factors may be required for the formation of an enzymatically active dimeric JHE (Chapter 6).Finally, following expression of specific genes gives us accurate information about the initiation of a developmental program. In long-day beetles, the JHE gene is switched off and the JH titer increases, leading to reproduction with vitellogenin expression. In contrast, short-days result in switching on of the JHE gene and a low JH titer. This induces the alternative developmental program, diapause, with the expression of diapause proteins. How signal transduction occurs from photoperiodic perception to gene activity is unknown yet (Chapter 7).</p

The use of attenuated isolates of Pepino mosaic virus for cross-protection

Pepino mosaic virus (PepMV) has recently emerged as a highly infectious viral pathogen in tomato crops. Greenhouse trials were conducted under conditions similar to commercial tomato production. These trials examined whether tomato plants can be protected against PepMV by a preceding infection with an attenuated isolate of this virus. Two potential attenuated isolates that displayed mild leaf symptoms were selected from field isolates. Two PepMV isolates that displayed severe leaf symptoms were also selected from field isolates to challenge the attenuated isolates. The isolates with aggressive symptoms were found to reduce bulk yields by 8 and 24% in single infections, respectively. Yield losses were reduced to a 0–3% loss in plants that were treated with either one of the attenuated isolates, while no effects were observed on the quality of the fruits. After the challenge infection, virus accumulation levels and symptom severity of the isolates with aggressive symptoms were also reduced by cross-protection. Infection with the attenuated isolates alone did neither affect bulk yield, nor quality of the harvested tomato fruits

Domain-specific expertise of chemistry teachers on context-based education about macro-micro thinking in structure-property relations

This study aims to determine and describe the new domain-specific expertise of experienced chemistry teachers in teaching an innovative context-based unit about macro– micro thinking in structure–property relations. The construct of ‘teachers’ domain-specific expertise’ was used to analyse the new repertoire chemistry teachers need to acquire to teach a context-based unit and achieve the intended effects of the curriculum innovation. A phenomenological approach of exploration and verification of teachers’ new repertoire resulted in the description of seven themes. These themes were related to the new aspects of the unit: the context-setting, the teacher’s role and the new content. In addition, the results show that the theoretical framework of teachers’ domain-specific expertise is feasible for the analysis and description of their new repertoire in the domain of teaching a contextbased unit. Further research is necessary to explore the use of the framework from the perspective of teachers’ professional development, where affective components in teachers’ learning processes play an important role

Expression of the juvenile hormone esterase gene in the Colorado potato beetle, Leptinotarsa decemlineata : Photoperiodic and juvenile hormone analog response

Metamorphosis and reproduction in insects are controlled by juvenile hormone (JH). One of the factors, which regulate the JH titer in the hemolymph, is the activity of juvenile hormone esterase (JHE). JHE from the Colorado potato beetle, Leptinotarsa decemlineata, consists of two 57kDa subunits. In this study, the JHE-cDNA was used as a probe to examine where and when the gene is transcribed as well as how gene expression responds to photoperiodic treatment and to topical application with a JH analog, pyriproxyfen. JHE transcripts were almost exclusively found in RNA extracts from fat body tissue in both larvae and adults. JHE-mRNA levels in the fat body correlated positively with levels of JHE activity in the hemolymph. In the last larval instar, high levels of JHE-mRNA were found in the feeding stage. In adults, reared under short-day conditions, JHE-mRNA levels were high between day 2 and day 9, which correlated with high JHE activity in the hemolymph. During these conditions, the JH titer decreases in preparation for pupation and diapause, respectively. The JHE-mRNA levels and JHE activity in the hemolymph were higher in short-day than in reproductive long-day adults. If the JH analog pyriproxyfen was applied to animals of the last larval instar on day 0 or day 3, JHE gene expression was enhanced. In contrast, if pyriproxyfen was applied to short-day adults on day 1 or day 4, the mRNA levels and the JHE activity in the hemolymph were suppressed to levels similar to those found in long-day adults. Thus, transcription of JHE is dependent on developmental stage, tissue, photoperiod and the level of its substrate J

Cloning and sequence analysis of cDNA encoding a putative juvenile hormone esterase from the Colorado potato beetle.

In the Colorado potato beetle, Leptinotarsa decemlineata, reproduction and diapause are mediated by the juvenile hormone (JH) titer in the hemolymph. This titer is controlled by JH synthesis in the corpora allata and by JH degradation. The main pathway of JH degradation is by JH esterase in the hemolymph. The native JH esterase appeared to be a dimer consisting of two 57 kDa subunits (Vermunt et al., 1997). The 57 kDa subunit of JH esterase was digested with endoproteinase Lys-C and the digestion products were separated by reversed phase HPLC. Three different peptides were collected and sequenced. The amino acid sequence of one peptide showed high similarity to fragments of other insect esterases. Based on the amino acid sequence of these peptides, degenerate primers were constructed for RT-PCR. A PCR product of 1.3 kb was obtained and sequenced. This product was used to screen a cDNA library for a complete cDNA copy and to analyze the messenger RNA from larvae and adult beetles. The size of the messenger RNA was 1.7 kb. The complete amino acid sequence of the protein was deduced from the nucleotide sequence of overlapping clones from a cDNA library and a 5&unknown;RACE product. An open reading frame (ORF) of 1545 base pairs encoded a 57 kDa protein with a predicted pI of 5.5. The ORF contained the sequences of the three peptides. It showed no significant homology to other proteins present in databases, but it did contain several functional esterase motifs

Strategies to support teachers’ professional development regarding sense-making in context-based science curricula

The aim of this study is to develop more understanding about strategies to support teachers' professional development in curriculum innovations, in which pedagogy and content change simultaneously compared to the conventional curriculum. A pre‐existing framework, including strategies for professional development, was adapted, implemented, and evaluated from the perspective of teachers' sense‐making in teaching context‐based science curricula. This framework guides the design of activities that support teachers' development in three new aspects of teaching context‐based science units: setting a context in class, performing a new teaching role, and teaching new content. In a case study, six teachers in secondary education participated in a professional development program based on the adapted framework. A qualitative inner‐case analysis was conducted to describe teachers' sense‐making during the program, in terms of the categories “assimilation,” “accommodation,” “toleration,” and “distantiation.” Results showed that teachers participating in the professional development program successfully assimilated and accommodated all three aspects; however, the process of teachers' sense‐making of the new content followed a different path compared to the processes of the other aspects. The relation between these results and the adapted framework are discussed to retrieve strategies for planning professional development programs to support teachers in curriculum innovations